Pro-inflammatory cytokines contribute to the decline in islet function during the development of diabetes. Cytokines can disrupt insulin secretion and calcium dynamics; however, the mechanisms underlying this are poorly understood. Connexin36 gap junctions coordinate glucose-induced calcium oscillations and pulsatile insulin secretion across the islet. Loss of gap junction coupling disrupts these dynamics, similar to that observed during the development of diabetes. This study investigates the mechanisms by which pro-inflammatory cytokines mediate gap junction coupling. Specifically, as cytokine-induced NO can activate PKC␦, we aimed to understand the role of PKC␦ in modulating cytokine-induced changes in gap junction coupling. Isolated mouse and human islets were treated with varying levels of a cytokine mixture containing TNF-␣, IL-1␤, and IFN-␥. Islet dysfunction was measured by insulin secretion, calcium dynamics, and gap junction coupling. Modulators of PKC␦ and NO were applied to determine their respective roles in modulating gap junction coupling. High levels of cytokines caused cell death and decreased insulin secretion. Low levels of cytokine treatment disrupted calcium dynamics and decreased gap junction coupling, in the absence of disruptions to insulin secretion. Decreases in gap junction coupling were dependent on NO-regulated PKC␦, and altered membrane organization of connexin36. This study defines several mechanisms underlying the disruption to gap junction coupling under conditions associated with the development of diabetes. These mechanisms will allow for greater understanding of islet dysfunction and suggest ways to ameliorate this dysfunction during the development of diabetes.Diabetes is characterized by a progressive decrease in function and mass of ␤-cells, which comprise the majority of cells in the islets of Langerhans (1). Pro-inflammatory cytokines have been implicated as mediators of ␤-cell death in both type 1 diabetes (T1D) 2 and type 2 diabetes (T2D) (2-4). However, pro-inflammatory cytokines also play a role in causing ␤-cell dysfunction early in disease progression (3, 5). In T1D, high levels of pro-inflammatory cytokines, including tumor necrosis factor-␣ (TNF-␣), interleukin-1␤ (IL-1␤), and interferon ␥ (IFN-␥), are released by immune cells, such as CD4 ϩ and CD8 ϩ T-cells and macrophages, which infiltrate the pancreas (3, 6). In T2D, adipocyte stress resulting from obesity can lead to secretion of low levels of circulating TNF-␣ from activated macrophages in adipose tissue; whereas elevated free fatty acids and/or hyperglycemia can also lead to local release of IL-1␤ in the islets (4, 7). Although the mechanisms of cytokine-induced cell death in diabetes are well characterized, cytokine-induced islet dysfunction is poorly understood.In vitro, the pro-inflammatory cytokines TNF-␣, IL-1␤, and IFN-␥ work synergistically (8) to induce islet dysfunction and disrupt insulin secretion (9, 10). The effect of pro-inflammatory cytokines on the ␤-cell is thought to be mediated in part by...

Key points The pancreatic islets of Langerhans maintain glucose homeostasis through insulin secretion, where insulin secretion dynamics are regulated by intracellular Ca2+ signalling and electrical coupling of the insulin producing β‐cells in the islet. We have previously shown that cytokines decrease β‐cell coupling and that compounds which increase cAMP can increase coupling. In both mouse and human islets exendin‐4, which increases cAMP, protected against cytokine‐induced decreases in coupling and in mouse islets preserved glucose‐stimulated calcium signalling by increasing connexin36 gap junction levels on the plasma membrane. Our data indicate that protein kinase A regulates β‐cell coupling through a fast mechanism, such as channel gating or membrane organization, while Epac2 regulates slower mechanisms of regulation, such as gap junction turnover. Increases in β‐cell coupling with exendin‐4 may protect against cytokine‐mediated β‐cell death as well as preserve insulin secretion dynamics during the development of diabetes. Abstract The pancreatic islets of Langerhans maintain glucose homeostasis. Insulin secretion from islet β‐cells is driven by glucose metabolism, depolarization of the cell membrane and an influx of calcium, which initiates the release of insulin. Gap junctions composed of connexin36 (Cx36) electrically couple β‐cells, regulating calcium signalling and insulin secretion dynamics. Cx36 coupling is decreased in pre‐diabetic mice, suggesting a role for altered coupling in diabetes. Our previous work has shown that pro‐inflammatory cytokines decrease Cx36 coupling and that compounds which increase cAMP can increase Cx36 coupling. The goal of this study was to determine if exendin‐4, which increases cAMP, can protect against cytokine‐induced decreases in Cx36 coupling and altered islet function. In both mouse and human islets, exendin‐4 protected against cytokine‐induced decreases in coupling and preserved glucose‐stimulated calcium signalling. Exendin‐4 also protected against protein kinase Cδ‐mediated decreases in Cx36 coupling. Exendin‐4 preserved coupling in mouse islets by preserving Cx36 levels on the plasma membrane. Exendin‐4 regulated Cx36 coupling via both protein kinase A (PKA)‐ and Epac2‐mediated mechanisms in cytokine‐treated islets. In mouse islets, modulating Epac2 had a greater impact in mediating Cx36 coupling, while in human islets modulating PKA had a greater impact on Cx36 coupling. Our data indicate that PKA regulates Cx36 coupling through a fast mechanism, such as channel gating, while Epac2 regulates slower mechanisms of regulation, such as Cx36 turnover in the membrane. Increases in Cx36 coupling with exendin‐4 may protect against cytokine‐mediated β‐cell dysfunction to insulin secretion dynamics during the development of diabetes.

Pulmonary hypertension (PH) is a progressive disease that is characterized by a gradual increase in both resistive and reactive pulmonary arterial (PA) impedance. Previous studies in a rodent model of PH have shown that reducing the hemodynamic load in the left lung (by banding the left PA) reverses this remodeling phenomenon. However, banding a single side of the pulmonary circulation is not a viable clinical option, so -using in silico modeling- we evaluated if the banding effect can be re-created by replacing the proximal vasculature with a synthetic PA. We developed a computational model of the pulmonary circulation by combining a 1-D model of the proximal vasculature with a 0-D line transmission model to the 12th generation. Using this model, we performed 4 simulations: (1) Control; (2) PH; (3) PH with a stenosis in the left PA; and (4) PH with proximal vessel compliance returned to Control levels. Simulations revealed that vascular changes associated with PH result in an increase in PP and WSS, relative to controls, in the distal circulation. Banding the left PA reduced distal PP and WSS in the left lung. Returning the proximal compliance to Control levels achieved the same effect (as banding the left lung) in both lungs, but also restored the shape of the pressure waveform similar to Control. In conclusion, we present a computational framework to estimate hemodynamic unloading in the distal circulation of rats after LPA banding and show that this can be done by restoring proximal vessel compliance.

Pancreatic islets regulate glucose homeostasis through insulin secretion. β‐cells are connected by gap junctions, which regulate depolarization, calcium signaling and insulin secretion. In diabetes, pro‐inflammatory cytokines contribute to β‐cell mass depletion and dysfunction. Low levels decrease Gap Junction Coupling (GJC) in the islet, disrupting calcium signaling and insulin secretion dynamics. Cyclic adenosine monophosphate (cAMP), elevated by a Type 2 Diabetes treatment called Exendin‐4, has been shown to increase GJC. Increased cAMP acts via protein kinase A (PKA) and exchange protein activated by cAMP 2 (Epac2) to regulate GJC in other systems; however, this has not been shown in pancreatic islets. We hypothesized Exendin‐4 can overcome cytokine induced decreases in GJC through PKA and Epac2 dependent mechanisms.To test this, isolated mouse islets were cultured overnight with a cocktail of pro‐inflammatory cytokines, 10nM Exendin‐4 and activators and inhibitors of PKA and EPAC2. Changes in GJC were quantified with Fluorescent Recovery After Photo‐bleaching, and calcium signaling was measured with calcium imaging of Fluo‐4.Cytokine+Exendin‐4 increased GJC compared to the cytokine treatment alone. PKA inhibitor decreased the Exendin‐4 recovery and had higher GJC than the cytokine treatment. PKA activator also recovered cytokine induced GJC decreases. Epac2 had little effect on recovery.These results suggest that Exendin‐4 is acting through PKA to overcome cytokine induced decreases in GJC while EPAC2 has minimal effect. Future work is needed to determine how PKA increases GJC via Cx36 in this system. This work will be important for targeting decreased GJC in pre‐diabetic patients.